Erythroid Krupple Like Factor (EKLF) is the only known erythroid-specific transcription factor, and this zinc finger protein is essential for correct gamma to beta-globin gene switching. Although EKLF was discovered a number of years ago and much has been learned about its function, large gaps still exist in our understanding of the way in which this critical transcription factor regulates globin gene expression in vivo. A more detailed understanding of the mechanism of EKLF action will provide insights into globin gene regulation and will suggest novel methods for maintaining or reactivating human fetal globin gene expression in patients with hemoglobinopathies such as beta-thalassemia and sickle cell disease. A novel model for globin gene switching that is consistent with the EKLF binding profile described in this proposal is as follows. EKLF forms different complexes in primitive and definitive erythroid cells and these different complexes direct globin gene switching. In primitive cells, the EKLF complex binds to ?y2, ?h1 and ?-major/minor globin gene CACCC boxes equivalently;however, ?y2 and ?h1 genes are preferentially expressed because these genes are more proximal to the LCR. In definitive cells, a new EKLF complex is formed (perhaps as a result of the 3-fold increase in EKLF protein levels) and this complex binds specifically to the adult gene CACCC boxes. In this case, the more distal adult genes preferentially interact with the LCR and are consequently expressed at high levels. The central hypothesis of this proposal is that EKLF complexes in primitive and definitive erythroid cells are different and that these differences are essential for correct globin gene switching. The Specific Aims designed to test this hypothesis are: 1. To affinity tag the endogenous EKLF transcription factor gene with several Tandem Affinity Purification (TAP) tags and to produce mice that are homozygous for this modification. 2. To purify protein complexes from primitive and definitive erythroid tissues of the TAP tagged mice and to define the components of these complexes by mass spectrometry. 3. To determine the role of EKLF complexes in globin gene switching. These experiments will provide new mechanistic insights into globin gene switching and should provide a foundation for the development of new therapies for hemoglobinopathies such as sickle cell disease.